Interaction between noradrenergic and cholinergic systems in the rat brain: Behavioural function in locomotor activity

INTERACTION BETWEEN NORA~RENERGI~ AND CHOLINERGIC SYSTEMS IN THE RAT BRAIN: BEHAVIOURAL FUNCTION IN LOCOMOTOR ACTIVITY S. 1. MASON and H. C. FIBIGER Division of Neurological Sciences. Department of Psychiatry, University of British Columbia Vancouver. B.C.. V6T IWS, Canada Ahatraet-Anticholinergic drugs such as scopolamine and atropine induce a mild locomotor stimulation when given intraperitoneally to rats. This effect is usually ascribed to interaction between dopaminergic and cholincrgic transmission in the striatum or nucleus accumbens. However. an interaction of acetylcholine with noradrenergic systems is also apparent from biochemical data and the results reported here indicate that at least part of the locomotor activity induced by scopolamine or atropinc involves a noradrenergic component. Depletion of forebrain noradrenaline by injection of 4 fig of the selective neurotoxin Ghydroxydopamine into the dorsal bundle was found to potentiate the locomotor activation induced by various doses of scopolamine or atropine. This was a central effect since methylscopolamine, which does not pass the blood-brain barrier. failed to induce locomotor activity and was not affected by the noradrenergic lesion. The noradrenergic interaction was restricted to cholinergic drugs since locomotor activity induced by the indirect dopamine agonist amphetamine was not affected by noradrenaline depletion. These studies show that the interaction between noradrcnergic and cholinergic transmission, which has previously been indicated by biochemical analysis. influences behaviour and they also cast some light on the functions of the central noradrenergic system itself.

DRUGS that reduce cholinergic function such as Scopolamine and atropine, which block muscarinic cholinergic receptors in the central nervous system, are found to induce a mild locomotor stimulation when administered to rats (CAMPBELL.LYTLE & FIBIGER, 1969). Conversely. drugs which enhance cholinergic function. either by direct muscarinic agonist action, such as pilocarpine or arecoline. or by indirect facilitation of cholinergic transmission by preventing transmitter catabolism, such as physostigmine, are found to induce an opposite state of catalepsy or tonic immobility (ZETLUI, 1968). These effects have typically been atttributed to an interaction with dopaminergic systems (BARTHOL~~~, STADLER& LLOYD. 1975), particularly in the striatum or nucleus accumbens (KELLY, SEVIOUR& IVERSEN,1975), and extensive biochemical evidence indicates that such an interaction almost certainly occurs. However, recent evidence suggests that acetylcholine may also interact with another catechoiamine, noradrenaline, in the central nervous system. A cholinergic-noradrenaline interaction is well documented in the peripheral nervous system (WESTFALL,1977) and biochemical evidence suggests that such also occurs in the central nervous system (CNS). Thus. cholinergic agonists are found to deplete noradrenaline Ieveis in various brain regions (Grrsso~. KARCZMAR& BAR=, 1972; t974), to influence noradrenaline release from in 1Qrr0prep arations (WESTFALL.1974~: 1974b) and to increase noradrenaline release in ciuo in man as judged by Abbre~~af~un: 6-OHDA. ~hydroxydopamine.

meta~iite con~ntratio~ in ~ebr~pin~ fluid (DAVIS, HOLLISTER,C&BODWIN% GORDON, 1977). As yet no behavioural function has been suggested for this noradrenaline-acetylcholine interaction. Noradrenergic systems have been suggested to be involved in a variety of behaviours, including blood pressure (WARD & GUNN, 1976), sleep (Jouvrr, 1%9), learning (CROW. 1973) and extinction (MASON & Ivtmm, 1975). Occasionai suggestions of a role for noradrenaline in locomotor activity (CORRODI, FuXE, LIUNGDAHL& OGREN, 1970; STROMBERG8s SVENSSON, 19711, probably as a consequence of its effects on arousal (L1DBRlNK, 1974; LIDBR~M(& FUXE, 1973) have been made. We have studied here whether depletion of forebrain noradrenaline using the selective neurotoxin, 6-hydroxydopamine (60HDA) (IJRETSKY & IVERSEN,1969) potentiates the locomotor stimulant effects of anticholinergic drugs without any change in either spontaneous locomotor activity or activity induced by d-~phet~ine. EXPERIMENTAL PROCEDURES Surgical Ten male albino Woodlyn rats weighing about 3oOg at the time of operation were anaesthetized with sodium pentobarbitone (Nembutal, SOmgikg i.p.). positioned in a stereotaxic apparatus (David Kopf Instruments) and two holes drilled in the skull. A 34gauge cannula was lowered bilaterally to the following co-ordinates: AP + 2.6mm from the interaural line, ML + 1.1 mm from the midline suture at the bregma, and DV + 3.7mm from the inter-

aural line with the animal’s head in the plane of 517

K~NIG

518

5 T. MASON and H. C. FIBIGER

h KLIPPEL(1963). This corresponds to the course of the ascending noradrenergic fibres of the dorsal bundle in the mesencephalon (UNGERSTEDT, 1971). Four micrograms ol 6-OHDA [weight expressed as free base of 6-OHDA HBr. Regis Chemicals) dissolved in 2~1 of 0.9% saline with 0.2 mglml ascorbic acid antioxidant was infused at the rate of 1pi per min for 2min and the cannula left in for a further minute to permit diffusion of the drug. The control animals received infusion of the same volume of salineascorbate solution. The skin was sutured and 2 weeks allowed for complete anterograde degeneration of the terminals before behavioutal testing started (Ross & REIS, 1974).

removal of the vertebrae using rongeurs. About 5cm of the rostra1 cord was taken for assay. These regions were then homogenized in 0.1~ perchloric acid and assayed for endogenous catechotamines by a spectrophotofluorometric method (MCGEER& MCGERR,1962). This served to confirm that the 6-GHDA manipulation had produced the expected pattern and extent of amine depletion. The brain stem region containing the injection site was kept in IO”,, formalin solution for at least I week and then sectioned on a freezing microtome at 50pm intervals and stained with Cresyl Violet to reveal the Nissl substance. Sratisticai

Analysis of variance (WINER,1962) was applied to the untransformed locomotor data using a design with Locomotor activity was tested in circular photocell activity cages (BRS/LVE Instruments, Beltsville, Maryland) measuring 61 cm in diameter and being transected by three photocell beams in the horizontal plane and another three at right angles to the first in the horizontal plane. Interruption of any beam activated an electromechanical counter located at a distance from the cage. Photocell beam interruptions were cumulated over 10 mm and then printed out of an automatic print-out counter (BRSLVE). The cages were positioned in a constant illumination and white noise was present in the environment. Testing took place from 10 a.m. to 4 p.m.’ with animals being on ad lib food and water in their home cages. One hour of habituation was allowed to the apparatus and then the drug, dissolved tn physiological saline, was injected intraperitoneally, and the anima1 replaced in the cage for a further 2 h during which time photoceli beam interruptions were recorded every IOmin. Animals were housed under a 12-h non-reversed light-dark cycle. The following drugs were used: scopolamine hydrobromide (Sigma), atropine nitrate (Sigma), d-amphetamine sulfate (Smith, Kline and French) and methylscopolamine hydrobromide (Sigma). Doses of 0.5. 1.0, 2.0 and 4.0mg/kg scopolamine, 2, 10 and 50mg/kg of atropine, I m&kg methylsco~~amine and I mgjkg d-amphetamine were used. Doses are in terms of the aforementioned salts.

repeated measures on the factor, time, for each individual drug dose, including saline injections. Conventional 5”:, level of significance was required (two-tailed).

Eiochemicai

the ascending noradrenaline

At the completion of behavioural testing the animals were killed by cervical fracture and the brains rapidly removed and dissected on ice into the following regions; hippocampus-cortex. hypothalamus, striatum. cerebellum and spinal cord. The brain was placed dorsal surface uppermost and the corpus callosum carefully split, the cortical hemispheres were then rolled forward and outwards revealing the hippocampus, which was blunt dissected from the cortical mass. The cortex was then repositioned and the brain rotated ventral surface uppermost. a coronal cut was made at the level of the mamillary bodies and the cerebellum removed from the posterior portion by blunt dissection of the~peduncles. The anterior portion of the brain was split along the midline and scissor cuts made from the anterior commissure in a vertical and horizontal direction to define the hypothalamus, which was then dissected free of the rest of the brain. The thalamus was discarded and the striatum separated from the overlying cortex by blunt dissection along the white matter of the corpus callosum. The remaining cortex dorsal to the rhinal fissure then constituted the cortical sample. The spinal cord was obtained by retractron of the musculature above the vertebrae and subsequent exposure of the cord after

Fig.

RESULTS

The post-mortem amine assays are shown in Table I and indicate that, after injection of &OHDA, severe and permanent depletion of hippocampal cortical noradrenaline to leas than S’?; of control values was brought about with considerable loss of hypothalamic noradrenaline as well. A small elevation was seen in noradrenaline concentrations in the cerebellum or the spinal cord, but there were no significant change in dopamine concentrations in the striatum. These data thus indicate that virtually complete destruction of the dorsal noradrenergic system (UNGERSTEDT,1971; LINDVALL& BJOORKLUND, 1974) was obtained with considerable, if subtotai, damage to the ventral noradrenergic system innervating the hypothalamus. Histological site in the m~encephalic course of fibre bundles is seen in l(a). A relatively small zone of ghosis was

The injection

TABLE 1. CATECHOLAMINE CONTENT OF BRAIN REGIONS IN RATS THAT HAD BEEN lNJECTED WffH ~~DROX~~PA~l~ IN THE WRSALBUNDLE

Region Noradrenaline Cortexhippocampus

Hypot~iamus Cerebellum Spinal cord Dopamine Striatum

Controls

264 + 6

6-OHDA lesioned

6tl

no

’

2240 +_77

590 f 87

26

219 2 12 255 & 6

271 + 8 307 + 12

124

11.570 + 1190

88

13,170 * 570

120

Control rats had received injection of saline, the others received 4pg of QOHDA into the dorsal bundle. Values are means with standard error of the mean in nanograms of amine per gram wet weight of tissue. The “0 column is the percentage of control concentrations remaining in lesioned tissues.

ibl FIG. 1. (a) Mcsenaphalic injection site showing small region of giiosis (arrow) at tip of cannula. Calibration bar is 1OOOpm. (b) Location of injection site relative to known course of fibres of the dorsal noradrenergic bundle. Abbreviations: LM, lemniscus medialis; SNR, substantia nigra: ip, interpeduncular nucleus. {et Higher power view of the site at the tip of injector cannula. Calibration bar is SO(fpm.

519

Noradrencrgic dorsal bundle and locomotor activity observed at the end of the cannula tract tarrow), which was located very close to the known trajectory of the dorsal noradrenergic bundle (Fig. 1b). A higher powered view of the small gliotic zone is shown in Fig. l(c). This gliotic zone was no greater than that seen fo~Iowing saline-ascorbic infusion in control rats.

The locomotor activity scores following intraperitoneal drug injections are shown in Fig. 3 for the 2 h after injection. No difference emerged between control and lesioned rats in spontaneous locomotor activity wtween groups F(l,lS) = 1.45, NS: interaction F(11,198) = 0.66, NS]. Following all doses of scopolamine and atropine used, significant locomotor stimulation was achieved relative to saline injection. The general pattern was that for the first hour no significant ditrerence was seen between control and lesioned rats but as the control locomotor activities declined during the second hour those of the lesioned rats failed to do so. This is shown in Fig. 2 for atropine and Fig. 3 for scopolamine. Thus, over the second hour the lesion4 animals were significantly more active than controls, as revealed by a significant group by time interaction. [Scopolamine F( 11,198) = 4.33, 2.55, 3.51 and 8.61 for the 0.5, 1.0, 2.0 and 4.Om~g dose respectively.) [Atropiae F(llJ98) = 2.03, 2.05 and 3.65 for the 2, 10 and 50mg/kg doses respectively.] The peripherally acting drug, methylscopolamine, which does not cross the blood-brain barrier, failed tb induce a locomotor stimulation compared to saline and no difference due to the lesion was seen [between groups F(1,18) = 0.13, NS and the interaction F(11,198) = 0.99, NSJ. The indirect dop~ine agonist, d-amphetamine, induced rather more locomotor stimulation than any dose of anticholinergics, but failed to affect the lesioncd group differentially between groups F(1.18) = 0.09, NS and the interaction F( 11,198) = 0.48, NS]. DISCUSSION No alteration in either the spontaneous or the amphetamine-induced locomotor activity was seen in the noradrenalinedepleted rats, despite forebrain noradrenaline levels being less than 5% of controls. This is similar to the unaltered spontaneous or amphetamine-induced locomotor activity in these animals reported previously (CR~E & IVERSEN,1975; ROBERTXZIS & F~BIGER,1975). Contrary to the general conclusion of noradrenergic non-involvement in psychomotor stimulant locomotor activity, a sign& cant alteration was seen in response to two cholinergic blocking agents, scopolamine and atropine. That the locomotor stimuiation induced by these antichoiinergic drugs, and the differential effect in the lesioned animals, was a central effect is shown by the failure of methylscopolamine (which does not cross the blood-brain barrier) to induce locomotor activity

521

or to affect the lesioned rats differentially. The alteration in chotinergic response was not, as is usually suggested, due to an interaction with dopaminergic systems (BARTHOLRJI, &ADLER& LLOYD,1975), since dopamine concentrations in the striatum, hypothalamus, amygdala or septum were not s~gn~~ntly changed by the lesion (FIBIGER & MASON,1978 and present results). Further, the effect of a drug which has been shown to act through dopaminergic systems (CREESE& IVERSEN,1975; ROBERTSet al., 1975; KELLY et ul., 1975), d-amphetamine, was unaltered. Although we only used a single dose of d-amphetamine in the present study, the above previous reports include extensive dose-response studies for d-amphe~~e in rats depleted of noradrenaline following placement of a lesion in the dorsal bundle, which show no change in the locomotor activity at any dose. Thus, our data provide prima facie evidence for a behaviouraily significant interaction between noradrenergic and cholinergic systems in the CNS. In other work (MASON,ROBERTS% FIBIG% 1978), we have shown that the effects of choiinergic agonists, arecoline and pilocarpine, which normally induce a hypoactive, cataleptic state (ZEXER, 1%8) are also influenced by noradrenergic mechanisms. Thus, catalepsy is greatly reduced or abolished by deptetion of forebrain noradrenaline levels, These cholinergic agonists are critically dependent on an intact noradrenaline system to exert their cataleptic actions and, in the case of the locomotor stimulation reported here, the action of anticholinergic drugs must also involve noradrenergic systems. In ttirn this has.implications for the function of the noradrenergic system per se. L~DBRINK(1974) has previously suggested a role of noradrenergic systems in arousal. Catalepsy and locomotor stimulation may be viewed as two extremes of an arousal continuum. This is in keeping with the previous reports of a noradrenergic involvement in locomotor activity (CORR~DI et af, 1970; STROMBERG& SVENSSON,1971). The dopaminergic systems, at least from our data, do not seem to share the same type of intera&ion with noradrenaline, since the dopaminergic stimulant d-amphet~ine was not influenced by our lesions. There are, however, reports of other aspects of dopamine function, such as stereotypy, which may be modulated by noradrenergic manipulations (DONAI.DSON. DOLPHIN, JEW, PYCOCK & MARSDEN,1978; PYCOCK,DONALDSON & MARSDEN,1975). Possible models fo account for the interaction between nor~~renergic and e~~~n~gic systems

A number of models may be postulated to account for functional interaction between acetylcholine and noradrenaline in anatomical terms. Perihar~u m~ei. It is known that the locus coeruleus receives cholinergic input from adjacent cell groups (PAPP & BOZS~K,1966; PAVLIN, 1966; SCHEBEL & SCHEBEL, 1973) and micrpiontophoretically applied acetylcholi~ excites these noradrenergic cells

522

S.

T.

MASON

and H. C.

(BIRD & KUHAR, 1977). Further, cholinergic agonists applied in this region are found to induce a state of atonia and catalepsy (AMATRUDA, BLACK, McKENNA,MCCARLEY& HOBS~N, 1975). According to this model, the increased terminal release of noradrenaline which would result from the cholinergic stimulation of the noradrenergic perikarya is a critical factor in producing the cataleptic response. Conversely, muscarinic antagonists would block the endogenous choiinergic activation of the noradrenergic neurons, thereby reducing normal noradrenaline release and hence resblting in motor stimulation, a behavioural state opposite to catalepsy. This model might predict that dorsal bundle lesions would by themselves increase locomotor activity. The fact that they do not, suggests that the anticholinergic drugs increase locomotor activity by actions on other neuronal systems in addition to the ascending noradrener-

FIBIGER

w

ATROPINE

50 mg/kg

400 w

CONTROLS

‘-*

06

~10

LESIONED n-10

120

(a)

4001

Zmg/kg

ATROPINE

I

TIME

o-0 CONTROLS . . .. 06

n-10

bins.)

FIG. 2. Locomotor activity in response to atropine nitrate injected intraperitoneally. Values are mean photocell beam interruptions of ten controt and ten laioned animals cumulated over 10 min for 2 consecutive hours. DB, dorsal

LESIONED n-10

bundle.

000 (min.%)

TIME

(b)

ATROPINE

10 mg/kg

600 D--O CONTROLS

F 5

soil

l

e-m 06

n=lO

;: 2 400

n-10

LESIONED

t

120 TIME

(mins.)

gic projections. That is, rather than being due to a decreased release of noradrenaline alone, motor stimulation induced by anticholinergic.drugs appears to be a combined result of a decreased release of noradrenaline and actions on other, presently unspecified systems. Similarly, although the ascending noradrenergic projections appear to play an essential role in muscarinic agonist-induced catalepsy, it is probable that concomitant cholinergic stimulation of other neuronal systems also contributes to this behaviour. Presynapric model. On the other hand, acetylcholine can affect the release of noradrenaline in terminal areas divorced from their cell bodies and tierent fibres (WESTFALL,1974a; 19746) with cholinergic innervation being prominent in a number of terminal areas innervated by noradrenaline such as the septum and hippocampus (WAS=, 1975). Additionally, localized intracerebral injection of atropine into either the septum or the hippocampus can induce locomotor activity (LEATON & RECH, 1972) and catalepsy is induced by injection of carbachol into these regions (MACLEAN.1957). This model would suggest that the action of acetyicholine on noradrenergic terminals leads to the release of noradrenaline. The action of the released noradrenaline is to reduce locomotor activity. Destruction of a large number of noradrenergic terminals by 6-OHDA injection would mean that the few remaining ones would have to release more noradrenaline than normal m order to maintain an unchanged spontaneous locomotor activity (which is observed

here).

The

cholinergic

input

to the

few

remaining terminals would assist in this by increasing

523

Noradrenergic dorsal bundle and locomotor activity (a)

SCOPOLAMINE

0.5

id)

WVkg

SCOPOLAMINE

4mWkg

60

0-u

CONTROLS

8-s

DB

n=lD 70

LESIONED

D

w

CONTROLS

.-‘.

06

n=lO

LESIONED n-10

n-10

TIME

(mind

FIG. 3. Locomotor

activity

in response to different

doses

of scopolamine. SCOPOLAYtNE

64

1 ma/k@

t

f

O-D CONTROLS

T

c

release of noradrcnatine. Blockade of this cholinergic input might render the few remaining noradrehergic terminals unable to maintain the usual level of locomotor activity and so a greater response to antichoiinergic drugs would be seen in the lesioned rats compared to controls. This enhanced sensitivity of the noradr~er~~ system, after lesions, to antichoiinergics is similar to that we11 proven for the dopaminergic system, following lesion, in its response to the dopamine synthesis inhibitor, a-methyl-ptyrosine. Following 6-OHDA lesion of the nigro-striatal dopaminergic system a dose of a-methyl-p-tyrosine which previousiy was ineffective. becomes able to disrupt behaviour (COOPER, Cm-r & WEEYE, 1974; COOPER, KONKOL & BREEE, 1978; BREISE& COOPER, 1975). We propose a similar mechanism of action for the enhanced response to anticholincrgic drugs seen here after depletion of noradrenaline stores. ~o~v~~e~ce modei. Both noradrenergic and choiinergic neurons may converge on a common postsynaptic cell and summate in activity in such a way that an intact noradrenergic input is needed for stimulation or blockade of the cholinergic receptor to be effective. No decision can be made between these models at the present but they are mentioned so as to be of heuristic value in directing future research. Additionally, our lesions result in severe depletion of noradrenaline in all terminal areas examined in the forebrain (hippocampus, cortex, hypothalamus, septum and amygdala; FBIGER % MASON, 1978) so caution must be exercised in specifying the location of the terminal areas on the presynaptic model. Nonetheless, the data reported here demonstrate for the first time a clear behavioural role of the biochemically indicated noradrenaline-acetylcholine interaction in the bruin and cast light on the functions of the noradrenaline systems per se. the

000 l -*

500 ,’

5 d: 400 I

DB

LESIONED n-10

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, F. ,* .* ‘.

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.

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.

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.

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524

S. T. MASONand H. C. FIBIGER REFERENCES

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16 Ocfober 1978)